The present invention relates to peptides which mimic carbohydrate epitopes (mimotopes) of Candida and to a vaccine comprising the peptide or polynucleotides encoding the peptide mimotopes or antibodies to the peptides and a method for the treatment of disseminated candidiasis due to infection by Candida albicans. 
Candida albicans is a fungus responsible for various forms of candidiasis, a condition which may be found in normal and immunocompromised patients, such as those with acquired immune deficiency syndrome. Humans and mice who are neutropenic are especially at risk of developing disseminated candidiasis (Denning, D. W., et al. 1992. Antifungal prophylaxis during neutropenia or allogeneic bone marrow transplantation: what is the state of the art? Chemotherapy 38 (suppl 1):43-49; Matsumoto, M. S., et al. 1991. Effect of combination therapy with recombinant human granulocyte colony-stimulating factor (rG-CSF) and antibiotics in neutropenic mice unresponsive to antibiotics alone. J. Antimicrob. Chemother. 28:447-453; Meunier, F. 1987. Prevention of mycoses in immunocompromised patients. Rev. Infect. Dis. 9:408-416; Meunier, F., et al. 1992. Candidemia in immunocom-promised patients. Clin. Infect. Dis. 14 (Suppl 1):S120-S125; and Van""t Wout, J. W. et al. 1989. Comparison of the efficacies of amphotericin B, Fluconazole, and Itraconazole against a systemic Candida albicans infection in normal and neutropenic mice. Antimicrob. Agents Chemother. 33: 147-151).
Several attempts have been made in the prior art to achieve immunostimulating compounds for the treatment of candidiasis as evidenced below.
U.S. Pat. No. 5,288,639 to Bernie et al. discloses the use of antibodies specific for stress proteins of C. albicans for the treatment of systemic candidiasis. Bernie et al. isolated a 47 kilo-dalton immunodominant antigen from C. albicans and found that serum from patients with systemic candidiasis reacts with the antigen. Monoclonal antibodies raised against the fungal stress proteins produced a 33% survival at 24 hours in animals challenged with a lethal dose of the C. albicans.
U.S. Pat. No. 4,397,838 to d""Hinterland discloses preparations of purified proteoglycans extracted from bacterial membranes. The proteoglycans serve as immuno-adjuvants and have an immunostimulating activity without being immunogenic themselves. They are useful in serving as adjuvants with ribosomal vaccines such as a vaccine containing the ribosomes of C. albicans. 
U.S. Pat. No. 4,310,514 to Durette et al. discloses immunologically active dipeptidyl 5-O, 6-O-acyl-2-amino-2-deoxy-D-glucofuranose derivatives. The compounds are used to delay the release of an antigen and stimulate the immune response of the host in conjunction with a vaccine. Compounds of Durette provide non-specific host protection against infectious organisms such as C. albicans. 
U.S. Pat. No. 4,315,913 to Durette discloses immunologically active dipeptidyl 2-amino-1,2-dideoxy-D-glucose derivatives. These derivatives are also useful as immunological adjuvants and themselves provide non-specific host protection against C. albicans. 
U.S. Pat. No. 4,368,910 to Shen et al. is directed to immunologically active dipeptidyl 4-O-6-O-acyl-2-amino-2-deoxy-D-glucose derivatives. These derivatives are indicated to be useful as immunogenic agents and vaccines and by themselves provide non-specific host protection against infectious organisms such as C. albicans. 
U.S. Pat. No. 4,323,560 to Baschang et al. is directed to phosphorylmuramyl peptides. The peptides are used to stimulate immunity. The compounds of Baschang et al. have been found to be inhibitive to infections caused by fungi such as C. albicans. 
U.S. Pat. No. 5,032,404 to Lopez-Berestein et al. disclose a liposomal agent for treating disseminated fungal infection in an animal. Because of the nature of polysaccharide fungal cell walls, it is expected that all medically important fungi activate complement. The patent indicated that there is a positive correlation between animals deficient in late-acting complement components and increased susceptibility to fungi such as C. albicans. The patent indicates that disseminated fungal infection can be treated with liposomal agent comprised of lipids, a polyene macrolide anti-fungal compound and cholesterol. Lipids can include phosphatidyl choline. Liposomes incorporate an effective amount of a polyene macrolide anti-fungal compound such as hamycins or lucensomycin, filipin, lagosin and natamycin.
U.S. Pat. No. 4,678,748 to Sutka et al. discloses a process for the production of the immunobiological preparations applicable in the diagnosis, prevention and treatment of Candida guilliermondii infections. Strains of C. guilliermondii are killed and used to formulate a vaccine.
Early attempts at obtaining compounds which provide non-specific host protection against C. albicans are generally in the form of immuno adjuvants used in conjunction with vaccines.
More specific vaccine approaches include targeting aspects of C. albicans pathogenesis. An important aspect of pathogenesis is adherence of C. albicans to host tissue. Discussion below provides an understanding of adherence as it relates to pathogenesis of disseminated candidiasis. C. albicans is an organism that may show considerable variability of certain characteristics. Genetics studies show that the organism is diploid, but apparently without the ability to undergo meiosis, yet it has impressive genetic variability between and within strains (Scherer, S. et al. 1990. Genetics of C. albicans. Microbiol. Rev. 54:226-241). Chromosomal aberrations unpredictably occur (Rustchenko-Bulgac et al. 1990. Chromosomal rearrangements associated with morphological mutants provide a means for genetic variation of C. albicans. J. Bacteriol, 172:1276-1283), and may be related to high frequency phenotypic (colony) changes in some strains (Soll, D. R. 1992. High-frequency switching in C. albicans. Clin. Microbiol. Rev. 5:183-203). Perhaps related to the genetic instability are findings that strains of C. albicans variably express cell surface antigens (Cutler, J. E., et al. 1994. Antigenic variability of C. albicans cell surface. Curr. Top. Med. Mycol. 5:27-47, and Martinez, J. P., et al. 1990. Wall mannoproteins in cells from colonial phenotypic variants of C. albicans. J. Gen. Microbiol. 136:2421-2432). Some of these antigens include putative virulence factors such as adhesions and enzymes (Cutler, J. E. 1991. Putative virulence factors of C. albicans. Ann. Rev. Microbiol. 45:187-218).
Studies on adherence properties of C. albicans are important in gaining an understanding of C. albicans interactions with its host. The ability to bind to mucus and epithelial surfaces likely plays a critical role in maintaining C. albicans at these locations. The fungus also shows adherence specificities for selected populations of splenic and lymph node macrophages (Cutler, J. E., et al. 1990. Characteristics of C. albicans adherence to mouse tissue. Infect. Immun. 58:1902-1908; Han, Y., et al. 1993. Binding of C. albicans yeast cells to mouse popliteal lymph node tissue is mediated by macrophages. Infect. Immun. 61:3244-3249; and Kanbe, T., et al. 1992. Evidence that C. albicans binds via a unique adhesion system on phagocytic cells in the marginal zone of the mouse spleen. Infect. Immun. 60:1972-1978), and extracellular matrix proteins (ECM) and endothelial cells (Filler, S. G., et al. 1991. C. albicans stimulates endothelial cell eicosanoid production. J. Infect. Dis. 164:928-035; Klotz, S. A. 1992. Fungal adherence to the vascular compartment: A critical step in the pathogenesis of disseminated candidiasis. Clin. Infect. Dis. 14:340-347; Mayer, C. L., et al. 1992. Technical report: C. albicans adherence to endothelial cells. Microvascular Res. 43:218-226; Rotrosen, D. et al. 1985. Adherence of Candida to cultured vascular endothelial cells: mechanisms of attachment and endothelial cell penetration. J. Infect. Dis. 153:1264-1274).
The fungal adhesions range in properties from hydrophilic to hydrophobic molecules (Hazen, K. C. 1990. Cell surface hydrophobicity of medically important fungi, especially Candida species, p. 249-295. In R. J. Doyle and M. Rosenberg (ed.), Microbial Cell Surface Hydrophobicity. American Society of Microbiology, Washington; Kennedy, M. J. 1988. Adhesion and association mechanisms of C. albicans. Curr. Top. Med. Mycol. 2:73-169) and all may be mannoproteins (8, 11). Both mannan and protein moieties may function as adhesions.
Some adhesions have integrin-like activity in that they act as receptors for mammalian proteins such as iC3b, fibronectin, laminin and fibrinogen; one adhesion has lectin-like activity; and a C3d receptor has been described (Bendel, C. M., et al. 1993. Distinct mechanisms of epithelial adhesion for C. albicans and Candida tropicalis. Identification of the participating ligands and development of inhibitory peptides. J. Clin. Invest. 92:1840-18492; Calderone, R. A., et al. 1991. Adherence and receptor relationships in C. albicans. Microbiol Rev. 55:1-20; Cutler, J. E. 1991. Putative virulence factors of C. albicans. Ann. Rev. Microbiol. 45:187-218; Gilmore, B. J., et al. 1988 An iC3b receptor on C. albicans: structure, function, and correlates for pathogenicity. J. Infect. Dis. 157:38-46; Klotz, S. A., et al. 1993. Adherence of Candida to immobilized extracellular matrix proteins is mediated by C. albicans calcium-dependent surface glycoproteins. Microbiol. 14:133-147). The surface of hydrophilic yeast cells of C. albicans has a fibrillar appearance both in vitro and in vivo (Hazen, K. C. et al. 1993. Surface hydrophobic and hydrophilic protein alterations in C. albicans. FEMS Microbiol. Lett. 107:83-88; Marrie, T. J., et al. 1981. The ultrastructure of C. albicans infections. Can. J. Microbiol. 27:1156-1164; and Tokunaga, M. et al. 1986. Ultrastructure of outermost layer of cell wall in C. albicans observed by rapid-freezing technique, J. Electron Microsc. 35:237-246).
A major component that makes up the fibrils on the cell surface of C. albicans and extends deeper into the cell surface appears to be the phosphomannoprotein (PMP). The cell surface is probably more complex than this, as additional proteins with relatively small amounts of carbohydrate may also be present (Hazen, K. C., et al. 1994. Hydrophobic cell wall protein glycosylation by the pathogenic fungus C. albicans. Can. J. Microbiol. 40:266-272). It is not clear, however, if these proteins differ from the major PMP or are the same proteins, but with a truncated version of the glycan portion.
The present inventors have overcome the deficiencies and inability of the prior art to obtain a vaccine against disseminated candidiasis by directing their attention to a composition comprising C. albicans adhesions.
Accordingly, an object of the present invention is to provide a vaccine for treatment of candidiasis comprising a pharmaceutically effective amount of peptides that are specific structural mimics (mimotopes) or epitopes specific to the mannan portion of the phosphomannan complex of Candida which elicits an immune response.
In a preferred embodiment the peptide is a nonapeptide with the amino acid sequence YRQFVTGFW(SEQ ID NO:8); where: Y, tyrosine; R, arginine; Q, glutamine; F, phenylalanine; V, valine; T, threonine; G, glycine; W, tryptophan.
In an alternative embodiment of the invention the peptide, which has a consensus amino acid sequence for peptides with reactivity to MAb B6.1, selected from the group consisting of, ArXXAr(Z)ZZArAr(SEQ ID NO:8); where: Ar, aromatic amino acid (F, W or Y); X, any amino acid; Z, equals S, (where S, serine), T or G; (Z), is S, T, or G which may or may not be present.
The invention also encompasses a vaccine wherein polynucleotide sequences encoding the peptide mimotope are delivered in an appropriate vaccine vector at pharmaceutical effective amounts for the treatment of candidiasis.
In a preferred embodiment of the invention, the polynucleotides are comprised of DNA coding for the peptide mimotopes and delivered in a DNA vaccine vector at pharmaceutical effective amounts for the treatment of candidiasis.
In an alternative embodiment of the invention, the polynucleotide vaccine comprises a DNA construct coding for a consensus amino acid sequence for peptides with reactivity to MAb B6.1, selected from the group consisting of, ArXXAr(Z)ZZArAr(SEQ ID NO:8); where: Ar, aromatic amino acid (F, W, or Y); any amino acid; Z, equals S (where S, serine), T or G; (Z), is S, T, or G which may or may not be present.
The invention also encompasses a vaccine wherein the mannan active portion comprises a composition structure selected from the group consisting of xcex2-1,2-linked straight chain tri, tetra- and penta-mannosyl residues in the acid labile part of the mannan portion of the phosphomannan complex.
Still another object of the invention provides a vaccine for treatment of disseminated and mucocutaneous Candidiasis comprising a pharmaceutical effective amount of an epitope of Candida Albicans comprising a beta 1,2-trimannose or acid stable epitopes that elicit an immune response.
The invention provides isolated protective antibodies for passive protection against hematogenous disseminated candidiasis and mucocutaneous candidiasis. The antibodies may be monoclonal antibodies specific for mannan epitopes in the acid stable portion of the mannan epitope and xcex2-1,2-linked tri, tetra- and penta-mannosyl residues in the acid labile part of the mannan portion of the phosphomannoprotein complex.
The invention also encompasses a vaccine wherein the mannan active portion comprises a composition structure selected from the group consisting of xcex2-1,2-linked straight chain tri, tetra- and penta-mannosyl residues in the acid labile part of the mannan portion of the phosphomannan complex.
Still another object of the invention provides a vaccine for treatment of disseminated and mucocutaneous Candidiasis comprising a pharmaceutical effective amount of an epitope of Candida Albicans comprising a beta 1,2-trimannose or acid stable epitopes that elicit an immune response.
The invention also encompasses a vaccine for treatment of disseminated candidiasis comprising a pharmaceutical effective amount of a peptide mimotope specific for Candida albicans epitopes, either xcex2 1,2-linked oligomannose or acid stable epitopes in the phosphomannan complex, or a vaccine comprised of polynucleotide sequences to code for said peptide, that elicit an immune response.
Still another embodiment provides a therapeutic composition for treatment of disseminated candidiasis comprising a pharmaceutical effective amount of passive humoral antibodies directed against a peptide mimotope specific for the xcex2 1,2-trimannose or others epitopes in the acid stable and acid labile regions of the mannan portion of the phosphomannan complex of Candida albicans that elicits an immune response. Also provided are isolated protective antibodies for passive protection against hematogenous disseminated candidiasis and mucocutaneous candidiasis.
The invention advantageously provides a method for the treatment of disseminated candidiasis and mucocutaneous candidiasis comprising administering an effective amount of the monoclonal antibodies of the invention to provide protection.
Still another embodiment provides a method for immunization against candidiasis comprising generating Candida albicans peptide mimotopes specific for phosphomannan complex-neutralizing antibodies.
Finally the invention provides a peptide mimotope specific to the mannan portion of the phosphomannan complex of candidiasis wherein said peptide has the amino acid sequence YRQFVTGFW(SEQ ID NO:1); where: Y, tyrosine; R, arginine; Q, glutamine; F, phenylalanine; V, valine; T, threonine; G, glycine; W, tryptophan, or function equivalents of said peptide. In a preferred embodiment the peptide has a consensus sequence of amino acids selected from the group consisting of, ArXXAr(Z)ZZArAr(SEQ ID NO:8); where: Ar, aromatic amino acid (F, W, or Y); X, any amino acid; Z, equal S (S, serine), T or G; (Z) is S, T or G which may or may not be present.
The above and other objects of the invention will become readily apparent to those of skill in the relevant art from the following detailed description, wherein only the preferred embodiments of the invention are shown and described, simply by way of illustration of the best mode of carrying out the invention. As is readily recognized the invention is capable of modifications within the skill of the relevant art without departing from the spirit and scope of the invention.